Isolation and purification of lung surfactant protein

ABSTRACT

Methods are provided for the isolation and purification of recombinant lung surfactant protein. The methods involve a multistep procedure including extraction with a C 1  -C 4  aliphatic alcohol and chromatographic purification steps which employ a C 1  -C 4  aliphatic alcohol as eluant. Purified proteins obtained using the disclosed isolation and purification steps are provided as well.

This application is a division of application Ser. No. 07/551,524, filedJul. 10, 1990, now U.S. Pat. No. 5,258,496.

TECHNICAL FIELD

This invention relates to lung surfactant proteins, and moreparticularly relates to novel methods for the isolation and purificationthereof.

BACKGROUND OF THE INVENTION

Respiratory distress syndrome (RDS), also known as hyaline membranedisease, is a major cause of morbidity and mortality of the prematurelyborn infant. RDS is believed to be caused primarily by a deficiency oflung surfactant--a lipid-protein mixture which coats the airspaces ofthe lung--thereby reducing surface tension and preventing airspacecollapse. The principal component of lungsurfactant--dipalmitoylphosphatidylcholine (DPPC)--was identifiedseveral years ago (Klaus et al., Proc Natl Acad Sci USA (1961) 47:1858;Avery et al., Am J Dis Child (1959) 97:517). It is believed thatadministration of lung surfactant to an individual having or at risk ofdeveloping RDS is a desirable therapy, and the literature disclosesvarious clinical studies of therapeutic administration of different lungsurfactant preparations.

Mammalian lung surfactant is a complex material containing primarilyphospholipids and associated proteins or apolipoproteins. The literaturecontains various lung surfactant protein preparations, including thosewith DPPC. Generally, these preparations include natural humansurfactant (purified from human amniotic fluid--Merrit et al. N Eng JMed (1986) 315:787); semisynthetic surfactant (prepared by combiningDPPC and high-density lipoprotein--Halliday et al. Lancet (1984) 1:476);animal lung surfactant (isolated by organic extraction of the whole lungor lavage fluid--Kwong et al. Pediatrics (1985) 76:585); and purifiedhuman surfactant apoproteins (SP-A, SP-B, and/or SP-C purified fromnatural sources or derived by recombinant DNA technology (Jobe et al. AmRev Resp Dis (1987) 136:1032, Glasser et al. J Biol Chem (1988)263:10326, PCT Publication WO 88/05820, and PCT Publication WO 90/01540,and which may be reconstituted with surfactant lipids (Revak et al. JClin Invest (1987) 81:826).

Significant progress has been made in the purification andcharacterization of the human surfactant apoproteins. However, differentinvestigators have observed variation in the number of peptides withapparent molecular masses between 5 and 18 kDa, and their nominalmolecular masses. Besides the molecular weight variations, researchershave experienced difficulty in separating the proteins isolated fromnative sources from their lipid components. Conventional lipidextraction procedures generally fail to separate the proteins completelyfrom the surfactant lipids. One procedure which addresses this problemis described by Arjomaa and Hallman (Ann Biochem (1988) 171:207-212).That process involves a two-step purification of a humanchloroform/methanol extract containing the SP4-6 peptide using a Sep PakFlorisil column, followed by reversed-phase high-pressure liquidchromatography (HPLC).

Recombinant production of surfactant proteins obviously does not requireseparation of protein from surfactant lipids. However, becausemicrobially produced surfactant proteins can be expressed at a fairlyhigh level, the high level of expression causes the recombinant proteinto precipitate intracellularly in the form of refractile bodies, whichdo require further isolation and purification. The problem addressed bythe present invention is how to safely and efficiently recoverrecombinant surfactant protein from the cell in a purified, renaturedform that is acceptable for clinical use.

Solvent systems used both in the extraction of lung surfactant proteinsand in subsequent chromatographic procedures typically involvecombinations of non-polar solvents and buffers such as ether/ethanol,ether/chloroform, and chloroform/methanol. Many of these non-polaragents are quite toxic; for example, chloroform has been shown to becarcinogenic in rats. The use of such solvents in the purification ofcompounds and compositions intended for pharmaceutical applicationstherefore raises product safety concerns about potential toxicity inhumans. It is, therefore, an object of the present invention to providea process for isolating and purifying recombinant lung surfactantprotein which avoids the use of chloroform-containing solvents andbuffers. The use of substantially non-toxic solvents permits one toemploy extraction and chromatographic procedures heretofore not used inthe purification of surfactant proteins.

BACKGROUND ART

PCT Publication No. WO88/05820, inventors Schilling et al., entitled"Recombinant Alveolar Surfactant Protein" and of common assignmentherewith, describes the complete coding sequences and amino acidsequences for canine and human 10K alveolar surfactant protein ("ASP").The reference also describes clones encoding variants of both the higherand lower molecular weight forms of human ASP.

PCT Publication No. WO90/00898 describes a process for extracting alipid and protein preparation from animal organs using a C₁ -C₁₀alkanol. The preparation may be used to treat respiratory distresssyndrome.

PCT Publication No. WO87/02037, inventors Taeusch et al., describes theisolation and characterization of certain alveolar surfactant proteins.

PCT Publication No. WO87/06943, inventor Whitsett, describes methods ofisolating an alveolar surfactant protein of 6,000 daltons and alsodescribes the coadministration of the aforementioned protein with lipidsfor the treatment or prevention of surfactant deficiency syndrome.

Whitsett et al. Pediatric Research 20(5):460-466 (1986), describe theisolation and characterization of various surfactant-associatedproteins. Whitsett et al. Pediatric Research 20(8):744-749 (1986), alsorelates to the surfactant-associated proteins and focuses specificallyon the lower molecular weight, i.e., 6,000 dalton, protein.

PCT Publication Nos. WO88/08849 and WO88/08850 describe processes forrecovering and purifying recombinant interleukin-2 (rIL-2) fromtransformed microorganisms using a sequence of solubilization andchromatographic procedures.

Tanaka et al. Journal of Lipid Research 27:475-485 (1986), present astudy of compositions containing synthetic lung surfactant protein andmixtures of synthetic lipids.

Tanaka et al. Chem Pharm Bull 31(11):4091-4099 (1983), Tanaka et al.Chem Pharm Bull 31(11):4100-4109 (1983), and Phizackerley et al. BiochemJ 183:731-736 (1979), all describe methods of isolating lung surfactantproteins which involve the use of chloroform as a solvent.

SUMMARY OF THE INVENTION

It is a primary object of the present invention to address theabove-mentioned needs in the art and to provide a process for isolatingand purifying recombinant lung surfactant SP-C (rSP-C) which avoids theuse of highly toxic solvents.

It is another object of the invention to provide such a method forisolating and purifying rSP-C which involves an extraction step with aC₁ -C₄ aliphatic alcohol or mixtures thereof.

It is still another object of the invention to provide such a processwhich involves separation of an SP-C fusion protein from a transformedmicroorganism, solubilization of inclusion bodies containing the fusionprotein, cleavage of the fusion protein, precipitation of the rSP-C fromthe post-cleavage mixture, and extraction of the rSP-C using a C₁ -C₄aliphatic alcohol or mixtures thereof.

It is yet another object of the invention to provide such a processwhich further involves one or more chromatographic purification stepseither instead of or following the extraction, wherein each of thechromatographic steps involves elution with a reagent composition thatcontains a C₁ -C₄ aliphatic alcohol or mixtures thereof.

In a further aspect of the invention, chromatographic purificationprocedures are provided which separate rSP-C monomer from mixtures oflung surfactant proteins containing rSP-C monomer and rSP-C multimers.

It is still a further object of the invention to provide purified rSP-Cobtained using the isolation and purification procedures described andclaimed herein.

In one aspect of the invention, the process for isolating and purifyingrSP-C from a transformed microorganism containing an expressed fusionprotein of rSP-C, comprises the steps of:

(a) disrupting the cell membrane and cell wall of the microorganism togive a mixture of (i) cellular components and (ii) inclusion bodiescontaining the fusion protein;

(b) separating the inclusion bodies containing the fusion protein fromthe cellular components;

(c) solubilizing the inclusion bodies;

(d) treating the solubilized inclusion bodies with a cleavage reagent,thereby cleaving the fusion protein derived from the inclusion bodies toyield a cleavage mixture containing rSP-C;

(e) precipitating protein containing rSP-C from the cleavage mixture toprovide a pellet that contains rSP-C; and

(f) extracting the rSP-C from the pellet with an extraction reagentcomposition which comprises a C₁ -C₄ aliphatic alcohol or an aqueoussolution of a C₁ -C₄ aliphatic alcohol.

In another aspect of the invention, step (f) in the just-describedprocess is followed by one or more chromatographic purification steps.In a preferred embodiment, chromatographic purification involveshydrophobic interaction chromatography on a cyanopropyl column and/or areversed-phase high-performance liquid chromatographic step. In bothcases, the eluant used contains a C₁ -C₄ aliphatic alcohol tospecifically solubilize the rSP-C.

In still another aspect of the invention, the aforementioned proceduresare used to purify rSP-B from a transformed microorganism containing anexpressed fusion protein of rSP-B.

In still other aspects of the invention, purified proteins obtainedusing the aforementioned processes are provided, as are individualextraction and chromatographic purification methods.

DETAILED DESCRIPTION OF THE INVENTION

A. Definitions and Overview:

As used herein, the term "rSP-C" refers to recombinant lung surfactantor lung surfactant-like polypeptides produced by a transformedmicroorganism and whose amino acid sequence is the same as, similar to,or substantially homologous to the native, relatively hydrophobicapproximately 3-5 k lung surfactant. Similarly, the term "rSP-B" refersto recombinant lung surfactant or lung surfactant-like polypeptideswhose amino acid sequence is the same as, similar to, or substantiallyhomologous to the native approximately 8 k lung surfactant. Examples ofsuch rSP-Bs and rSP-Cs are those described in PCT publicationsWO88/05820 and WO90/01540, of common assignment herewith. Thedisclosures of both these applications are incorporated herein byreference.

As used herein, the term "multimer" as applied to the rSP-C proteinrefers to dimers or larger aggregates of the rSP-C monomer.

Where "DTT" (dithiothreitol) is indicated as a preferred reagent in avariety of reagent compositions and procedures described herein, it willbe appreciated by those skilled in the art that any number ofalternative sulfhydryl reducing agent or agents could be used in itsplace.

The method of protein precipitation by water-miscible organic solventshas been employed since the early days of protein purification. Additionof an organic solvent to an aqueous extract containing proteins has avariety of effects which, in combination, can lead to proteinprecipitation. In general, the solvent used must be completelywater-miscible, unreactive with proteins, and effective to precipitatethe desired protein or proteins. Solvents that have been used to extractnative mammalian surfactant proteins include such non-polar solventsystems as ether/ethanol, chloroform, chloroform/methanol in variousratios such as 3:1. While these solvent systems have proved effectivefor various extraction and/or chromatographic procedures, they arepotential human carcinogens and thus present a risk, i.e., since usingthese solvents in purification procedures may result in somecontamination of the final product.

As shown herein, it has been discovered that recombinant SP-C can beeffectively solubilized with a number of lower aliphatic alcohols,employed either individually or in mixtures. Preferred alcohols compriseshorter carbon chains, as the longer-chain alcohols are more denaturingthan short-chain ones and may also be less soluble in water. The C₁ -C₄aliphatic alcohols useful herein include methanol, ethanol, n-propanol,isopropanol, n-butanol, isobutanol, and t-butanol. (Where it is statedherein that a C₁ -C₄ aliphatic alcohol may be used in extraction orchromatographic purification steps, it is to be understood that mixturesof such aliphatic alcohols may be used as well.)

B. Expression of the SP-C Fusion Protein:

The rSP-C is initially provided as a fusion protein expressed inbacterial cells. As is well-known, many eucaryotic proteins areincapable of being expressed in bacterial cells in any measurable yield,or, even if expressed in detectable quantities, are incapable of beingexpressed at commercially recoverable levels due to proteolysis of theforeign protein by the host. Small proteins such as SP-C appear to beespecially sensitive to such degradation. As explained in commonlyassigned application Ser. No. 07/391,277, filed 8 Aug. 1989, nowabandoned and incorporated herein by reference (published through thePCT as WO 90/01540 on 22 Feb. 1990), proteins which are incapable ofbeing expressed in high yield may be expressed as a fusion protein toincrease the level of expression. For purposes of the present invention,it is preferred that the SP-C be expressed as a fusion protein withchloramphenicol acetyltransferase (CAT), which is known as a selectablemarker and an easily assayed enzyme for the monitoring of efficiency ofboth eucaryotic and procaryotic expression (Delegeane, A. M., et al.Molecular Cell Biology, 7:3994-4002 (1987)).

The fusion proteins which one begins with in the isolation andpurification methods of the present invention are substantially asdescribed in the aforementioned patent application. The following is abrief summary of the fusion and expression process set forth in theaforementioned patent reference.

CAT encodes a 219 amino acid mature protein and the gene contains anumber of convenient restriction endonuclease sites (5'-PvuII, EcoRI,DdeI, NcoI, and ScaI-3') throughout its length to test gene fusions forhigh level expression. These restriction sites may be used for ease ofconvenience in constructing hybrid gene sequences.

Expression constructs using CAT can employ most of the CAT-encoding genesequence or a substantially truncated portion of the sequence encodingan N-terminal portion of the CAT protein linked to the gene encoding thedesired heterologous polypeptide. These expression constructs, whichdemonstrate enhanced levels of expression for a variety of heterologousproteins, utilize a number of varying lengths of the CAT protein rangingin size from 73 to 210 amino acids. The 73 amino acid CAT fusioncomponent is conveniently formed by digesting the CAT nucleotidesequence at the EcoRI restriction site. Similarly, the 210 amino acidCAT fusion component is formed by digesting the CAT nucleotide sequencewith ScaI. These, as well as other CAT restriction fragments, may thenbe ligated to any nucleotide sequence encoding a desired protein toenhance expression of the desired protein.

The reading frame for translating the nucleotide sequence into a proteinbegins with a portion of the amino terminus of CAT, the length of whichvaries, continuing in-frame with or without a linker sequence into theSP-C sequence, and terminating at the carboxy terminus thereof. Anenzymatic or chemical cleavage site may be introduced downstream of theCAT sequence to permit ultimate recovery of the cleaved product from thehybrid protein. Suitable cleavage sequences and preferred cleavageconditions will be described below.

To avoid internal cleavage within the CAT sequence, amino acidsubstitutions can be made using conventional site-specific mutagenesistechniques (Zoller, M. J., and Smith, M., Nucl Acids Res (1982)10:6487-6500 and Adelman, J. P., et al. DNA (1983) 2:183-193). This isconducted using a synthetic oligonucleotide primer complementary to asingle-stranded phage DNA to be mutagenized except for limitedmismatching, representing the desired mutation. Of course, thesesubstitutions would only be performed when expression of CAT is notsignificantly affected. Where there are internal cysteine residues,these may be replaced to help reduce multimerization through disulfidebridges.

Procaryotic systems may be used to express the CAT fusion sequence;procaryotic hosts are, of course, the most convenient for cloningprocedures. Procaryotes most frequently are represented by variousstrains of E. coli (e.g., MC1061, DH1, RR1, W3110, MM294, B,C600hfl,K803, HB101, JA221, and JM101); however, other microbial strains mayalso be used. Plasmid vectors which contain replication sites,selectable markers and control sequences derived from a speciescompatible with the host are used; for example, E. coli is typicallytransformed using derivatives of pBR322, a plasmid derived from an E.coli species by Bolivar et al. Gene (1977) 2:95. pBR322 contains genesfor ampicillin and tetracycline resistance, and thus provides multipleselectable markers which can be either retained or destroyed inconstruction of the desired vector.

In addition to the modifications described above which would facilitatecleavage and purification of the product polypeptide, the geneconferring tetracycline resistance may be restored to the exemplifiedCAT fusion vectors for an alternative method of plasmid selection andmaintenance.

Although the E. coli tryptophan promoter-operator sequences arepreferred, different control sequences can be substituted for the trpregulatory sequences. Commonly used procaryotic control sequences whichare defined herein to include promoters for transcription initiation,optionally with an operator, along with ribosome binding site sequence,include such commonly used promoters as the beta-lactamase(penicillinase) and lactose (lac) promoter systems (Chang et al. Nature(1977) 198:1056), the lambda-derived P_(L) promoter (Shimatake et al.Nature (1981) 292:128) and N-gene ribosome binding site, and the trp-lac(trc) promoter system (Amann and Brosius Gene (1985) 40:183).

Transformed microorganisms producing the fusion protein are grown in asuitable growth medium containing compounds which fulfill thenutritional requirements of the microorganism. Growth media willtypically contain assimilable sources of carbon and nitrogen, magnesium,potassium and sodium ions, and, optionally, amino acids, purine andpyrimidine bases, vitamins, minerals, and the like.

At the end of fermentation, the bacterial paste is collected by, e.g.,cross-flow filtration, centrifugation, or other conventional methods.The concentrated paste is preferably stored at a temperature below about-50° C., preferably about -70° C., until further rise.

C. Cell Disruption and Preparation of Inclusion Bodies:

Following concentration of the bacterial paste, the cell membranes andcell walls of the microorganisms are disrupted, either chemically, i.e.,with alkali or with a compound such as 1-octanol, enzymatically, e.g.,with lysozyme, or mechanically, i.e., using a commercially availablehomogenizer, as is well-known in the art. The end point of thedisruption step can be monitored by microscopy and/or by adding a dyesuch as Coomassie Blue and monitoring its absorbance at 595 nm, whichtypically increases with cell lysis. This process step should be carriedout for a time long enough to ensure that substantially all of the cellshave been disrupted, and that substantially no intact cells will becarried through to subsequent process steps.

After cell disruption, the insoluble fraction of the whole-cellhomogenate, containing inclusion bodies, is harvested by filtration,centrifugation, or the like. The inclusion body fraction is typically onthe order of 10% of the initial wet-cell weight, and enrichment for theCAT/rSP-C fusion protein is thus desirable. To remove contaminatingbacterial proteins, the inclusion body pellets are washed with a mediumwhich contains 1M guanidine hydrochloride and a protein-solubilizingagent such as Triton X-100, together with dithiothreitol (DTT), ethylenediamine tetraacetic acid (EDTA), and buffering agents. After washing,the inclusion bodies are pelleted by centrifugation and washed againwith the afore-mentioned medium. This washing procedure is important toachieve relatively high (50-70%) yields of rSP-C from the organic (C₁-C₄) aliphatic alcohol extraction step that follows later. This is dueto the fact that this procedure, by washing away part of thecontaminating bacterial protein, increases the proportion of inclusionbody protein that is CAT-rSP-C fusion protein (to about 40%). When 1Mguanidine·HCl is not included in the wash buffer, only about 25% of theinclusion body protein is CAT-rSP-C fusion, and the organic extractionyield falls to 20-30%. At this point, the inclusion bodies may, ifdesired, be frozen and stored.

Just prior to cleavage, the inclusion bodies are solubilized with abuffered medium containing on the order of 6M guanidine hydrochloridewith DTT and EDTA, followed by high-speed centrifugation. By "high-speedcentrifugation" is meant spinning the suspension in a centrifuge atabout 8,000-40,000×gravity (g), preferably about 10,000-20,000×g, for asuitable time period, depending upon volume, generally about 10 minutesto 72 hours.

D. Cleavage of the Fusion Protein:

The reagent and methods used in cleaving the fusion proteins willdepend, clearly, on the cleavage sequence incorporated into the fusionprotein at the outset. Cleavage sequences which may be used hereininclude, for example, those which may be cleaved following methionineresidues (cleavage reagent cyanogen bromide), glutamic acid residues(cleavage reagent endoproteinase Glu-C), tryptophan residues (cleavagereagent N-chlorosuccinimide with urea or with sodium dodecyl sulfate),and cleavage between asparagine and glycine residues (cleavage reagenthydroxylamine). For purposes of the present invention, cleavage withhydroxylamine is particularly preferred.

The inclusion bodies obtained in the previous step, in solubilized form,are treated with the selected cleavage reagent at ambient temperature.The reaction is allowed to proceed for as long as necessary to ensuresubstantially complete cleavage of the fusion protein.

E. Precipitation and Extraction of rSP-C:

After the cleavage reaction is complete, the reaction is stopped bydilution of the guanidine with buffered aqueous medium preferablycontaining a sulfhydryl reducing agent such as DTT (but not containingguanidine). This dilution causes precipitation of a proteinaceous masswhich includes the rSP-C. The pellet or paste collected upon subsequentcentrifugation is washed to remove extraneous matter and centrifugedagain. At this point, the rSP-C is specifically dissolved via extractionwith an extraction reagent composition which comprises a C₁ -C₄aliphatic alcohol or an aqueous solution of a C₁ -C₄ aliphatic alcohol.As noted above, the inventors herein have now found that such a reagentcomposition specifically dissolves rSP-C while allowing the remainingproteins to remain in a precipitated state. (The discovery that areagent composition containing a C₁ -C₄ aliphatic alcohol is useful inspecifically dissolving rSP-C was indeed surprising and unexpected inview of the fact that virtually all proteins are insoluble in loweralcohols.) Suitable C₁ -C₄ aliphatic alcohols are as identified above inthe section entitled "Definitions and Overview". Examples of preferredextraction reagent compositions include methanol, isopropanol, andaqueous solutions thereof. Examples of particularly preferred extractionreagent compositions include a 25% methanol/75% isopropanol mixture andaqueous solutions of 40%-100% isopropanol. Extraction may be repeated asnecessary to increase the yield of rSP-C obtained.

It is preferred that the compositions disclosed herein as extractionreagents and eluants containing a C₁ -C₄ aliphatic alcohol also containabout 0.001-100 mM acid, preferably a strong acid such as hydrochloric,trifluoroacetic, or phosphoric acid. This promotes the solubility of theprotein.

F. Ion-Exchange:

Following extraction, ion-exchange chromatography may be performed inorder to concentrate the rSP-C prior to size-exclusion chromatography.While any number of ion-exchange resins may be used in the initialchromatographic purification step, cation exchange resins such assulfopropyl cellulose ("SP-cellulose"), sulfonated Sepharose,carboxymethyl ("CM") cellulose, CM Sephadex, CM Sepharose, CM silica,and Trisacryl are preferred. Strong cation exchange resins such asSP-cellulose or sulfonated Sepharose are more preferable, while anSP-cellulose column is particularly preferred. As with all of thechromatographic purification steps used in the present process, theeluant, like the extraction reagent composition, comprises a C₁ -C₄aliphatic alcohol, or gradients of aqueous solutions of a C₁ -C₄aliphatic alcohol.

In carrying out the ion-exchange step, the extract of the precipitatedpost-cleavage protein mixture, is equilibrated with the prepared resin,preferably at least 1 hour and more preferably overnight at roomtemperature. The rSP-C is then eluted (preferably on the order of about6 hours at room temperature) with a reagent composition comprising a C₁-C₄ aliphatic alcohol, a buffer such as sodium acetate to increase pHand ionic strength, and a sulfhydryl reducing agent such asβ-mercaptoethanol. The volume of the eluant is preferably much smallerthan the volume of the extract, to effect marked concentration of therSP-C. The eluted rSP-C may, if desired, be further concentrated byrotary or flash evaporation.

G. Size-Exclusion Chromatography:

A size-separation step is carried out either in addition to or insteadof the aforementioned ion exchange step and is done using a sizingcolumn, as is well known in the art. The rSP-C may be concentrated byrotary or flash evaporation. The concentrated rSP-C is loaded onto theselected sizing column, and equilibrated prior to use preferably with aC₁ -C₄ aliphatic alcohol and 0.01-100 mM strong acid. A preferred resinfor use in the sizing column is Sephadex LH60. Chromatography on such acolumn removes low molecular weight buffer components that are notdesired in the final product, and assists in the separation of dimericand aggregated rSP-C from the monomeric final product. When the columnis run under preferred conditions, room temperature with a 75 cm longLH60 column, a pressure head of 60-65 cm, and an eluant reagentcomposition containing a lower aliphatic alcohol, as explained above,elution will occur after about 20 hours.

The preparation of rSP-C obtained from the sizing column is very pure,since rSP-C is soluble in the selected lower aliphatic alcohol while, asnoted earlier, virtually no other peptides in the post-cleavage mixtureare soluble under such extraction conditions.

H. Hydrophobic Interaction Chromatography Using a Cyanopropyl Column:

In another aspect of the invention, hydrophobic interactionchromatography is used to obtain pure rSP-C from a mixture of proteins,e.g., the entire post-cleavage protein mixture. Hydrophobic interactionchromatography can also be carried out following size exclusion toenhance the purity of the rSP-C obtained even further. The use of acyanopropyl resin in such a step has proved to be unexpectedly superiorrelative to other purification methods. A suitable cyanopropyl resin maybe obtained, for example, from Applied Biosystems (Foster City, Calif.).The cleavage mixture is preferably applied to the column in a bufferedcomposition containing the C₁ -C₄ aliphatic alcohol, guanidinehydrochloride, and dithiothreitol.

Development of the column is carried out with eluant compositionscontaining gradients of the C₁ -C₄ aliphatic alcohol. Preferably, thecolumn is run in the presence of low concentrations of acid, i.e., onthe order of 0.001-100 mM of a preferably strong acid such ashydrochloric, phosphoric, or trifluoroacetic acid, or the like, as notedabove with regard to extraction. Protein impurities elute early in thegradient while the rSP-C has been found to elute much later. The rSP-Cobtained using this process has been confirmed to be extremely pure(typically>90%) by PAGE and by N-terminal sequencing. Western blotanalysis also confirms these results.

I. Purification Using Reversed-Phase Resins:

In still another aspect of the invention, a method is provided forseparating rSP-C monomer from rSP-C multimers. The use of reversed-phaseHPLC for the separation of proteins is well-known; however, theresolution of protein or polypeptide mixtures on typical reversed-phasecolumns can be problematic. See, e.g., Practical Protein Chemistry: AHandbook, Ed. A. Darbre (N.Y.: John Wiley & Sons, 1986), at page 189.The present method combines the discovery that rSP-C monomer isselectively soluble in C₁ -C₄ aliphatic alcohols with the use ofreversed-phase, purely "hydrophobic interaction", matrices or resinssuch as C4, C8, C18, and phenyl. The use of C4 and C8 columns inconjunction with an HPLC process is particularly useful here, i.e., inthe separation of rSP-C monomer from rSP-C dimer in particular, sincethe dimer can remain associated with the monomer even after thepreceding chromatographic steps have been carried out.

As with the cyanopropyl column, elution of rSP-C in the reversed-phasesystem is preferably accomplished using gradients with increasingquantities of the C₁ -C₄ aliphatic alcohol. Again, a small quantity ofstrong acid is preferably present. Prior to loading the sample onto thereversed-phase column, the rSP-C obtained from the cyanopropyl columnmay be diluted with water or slightly acidic water. The diluted rSP-Cmay then be injected onto the reversed-phase column or dried,solubilized in a relatively high concentration of alcohol and dilutedagain in water or slightly acidic water. This procedure is believed tobe the first that enables clean separation of the rSP-C monomer from therSP-C dimer. The procedure may also be used on the post-cleavagemixture, but is more effective following the two chromatographicpurification steps just described.

Hydrophobic interaction chromatography and reversed-phase chromatographymay be carried out in either order. However, it is preferred thathydrophobic interaction chromatography be carried out first, followed bythe reversed-phase column to insure optimum separation of the rSP-Cmonomer from the dimer. In other embodiments of the invention, onechromatographic purification step is used and not the other, e.g., whereextremely pure rSP-C monomer is not required. In still other embodimentsof the invention, the chromatographic procedures may be carried outdirectly following cleavage of the fusion protein, i.e., withoutprecipitating or extracting the rSP-C.

J. Purification of rSP-B:

As noted above, "rSP-B" refers to recombinant lung surfactant or a lungsurfactant-like polypeptide which is substantially similar to the native8-10 k lung surfactant protein. Both SP-B derived from the surfactant ofbovine lungs and rSP-B isolated after bacterial expression have similarsolubilities in C₁ -C₄ alcohols such as isopropanol. In addition, thereversed-phase resins and specifically C4, C8, and phenyl, as well aslarge pore cyanopropyl resins, have been found to be useful in adsorbingSP-B. The elution characteristics for SP-B from these chromatographicsupports are very similar to those described for SP-C. Thus, while thepresent method has been described in terms of its utility with rSP-C.,it should also be useful in the isolation and purification of the lungsurfactant protein known as rSP-B using the procedure described andclaimed herein.

It is to be understood that while the invention has been described inconjunction with specific embodiments thereof, the foregoing descriptionas well as the examples which follow are intended to illustrate and notlimit the scope of the invention, which is defined by the appendedclaims. Other aspects, advantages and modifications within the scope ofthe invention will be apparent to those skilled in the art to which theinvention pertains.

Experimental

Assay for Lung Surfactant Activity

In vitro methods have been devised to assess the ability of lungsurfactant proteins to function by reducing surface tension (synonymouswith increasing surface pressure) to generate a film on an aqueous/airinterface. Studies using these methods have been performed on theisolated native 32K canine lung surfactant. (Benson et al. Prog Resp Res(1984) 18:83-92; Hagwood et al. Biochem (1985) 24:184-190).

EXAMPLE 1 Production, Isolation and Purification of Recombinant SP-C

Expression: The mature form of human SP-C was expressed as a fusionprotein with portions of the bacterial CAT protein. The surfactantprotein was joined to the carboxy terminus of the CAT sequence through ahydroxylamine-sensitive asparagine-glycine linkage. The CAT-surfactantfusion was expressed from the tryptophan promoter of the bacterialexpression vector pTrp233.

The plasmid was used to transform E. coli W3110 (ATCC accession no.27325) and selected for ampicillin resistance. The transformants weregrown in culture overnight at 37° C. in complete M9 medium containing M9salts, 2 mM MgSO₄, 0.1 mM CaCl₂, 0.4% glucose, 0.5% amino acids, 40μg/ml tryptophan, 2 μg/ml thiamine hydrochloride, and 100 μl/mlampicillin sulfate.

Homogenization: At the end of fermentation, the bacterial paste (2 kg inwet cell weight) was collected by centrifugation and stored at -70° C.until use. The frozen paste was thawed at 4° C. overnight andresuspended in 5 liters per kg cells of 20 mM Tris-HCl buffer, pH 8,containing 10 mM EDTA and 1 mM DTT. A Tekmar high-shear mixer was usedwith a G-450 probe. The cell suspension was approximately 20% solids.After a uniform suspension was achieved, cells were disrupted bycirculation through a Manton-Gaulin homogenizer equipped with a CD-30valve at 15,000 psi. The suspension was recirculated, with stirring, fora time equivalent to four complete passes of the suspension volumethrough the homogenizer. Samples of homogenate were monitored for celllysis by microscopy.

The undissolved fraction of the whole cell homogenate, containinginclusion bodies, was harvested by centrifugation at 5500×g for 30 minat 4° C. The inclusion body fraction was about 10% of the initial wetcell weight. The inclusion body pellet was enriched for the CAT-rSP-Cfusion protein by washing away contaminating bacterial proteins; thepellet was resuspended in buffer containing 1M guanidine·HCl, 20 mMTris-HCl , pH 8, 10 mM EDTA, 1 mM DTT and 1% v/v Triton X-100 bystirring at 50-55% capacity with the Tekmar mixer at 4° C. After 15 min,the inclusion bodies were pelleted by centrifugation as before, andwashed twice more with 10 volumes of buffer,. i.e., 10 l per kg ofinclusion body pellet. At this stage, inclusion body preparations werefrozen and stored at about -70° C. (stable for months).

Inclusion Body Solubilization and Clarification:

Washed inclusion bodies were thawed at 4° C. and solubilized with 10volumes of buffer (i.e., 10 ml per g wet weight) containing 6Mguanidine·HCl, 50 mM DTT, 50 mM EDTA, 20 mM Tris-HCl, pH 8, by stirringat 50-55% capacity at 4° C. for 30 min with the Tekmar mixer (G450 andG456 probes). The extract was clarified by centrifugation at 13,500×gfor 30 min at 4° C. The protein concentration of the extract wasdetermined using the BCA (bicinchoninic acid) assay (bovine serumalbumin as a standard), and then adjusted to 20 g/l.

Hydroxylamine Cleavage: CAT210/rSP-C fusion protein was cleaved withhydroxylamine. The cleavage reagent, freshly prepared immediately priorto use, was added to solubilized inclusion bodies at a 1:1 v/v ratio atambient temperature. The reaction was allowed to proceed at 25° C. in awater bath for 48 hr.

Cleavage reagent was prepared as follows: Hydroxylamine (NH₂ OH-HCl),140 g, was dissolved in 200 ml of 10.0M NaOH and 200 ml of 1.0M K₂ CO₃.Solid guanidine·HCl (573 g) was added and the solution stirred until itreturned to ambient temperature. The white precipitate that formed wasremoved by filtration through coarse scintered glass and 7.7 g of solidDTT were added with stirring. The pH was then adjusted to 10.0 using 10MNaOH and the volume brought to 1.0 l with pyrogen-free water. The bufferhad a final concentration of 2M hydroxylamine, 0.2M K₂ CO₃, 6Mguanidine·HCl, 50 mM DTT.

Precipitation and Extraction: After 48 hr, the reaction was stopped byfivefold dilution with 4° C. 20 mM Tris-HCl, pH 8, containing 20 mM DTT.After incubation for 30 min at 4° C., precipitating protein wascollected by centrifugation (15 min at 5500×g), washed twice with 0.4cleavage reaction volumes of the same buffer, centrifuged again anddrained inverted at 0° C. for 30 min.

Extraction of the precipitate was performed into isopropanol, using halfthe volume used in the cleavage reaction. Extraction was performed undernitrogen for 1-2 hours at room temperature on a magnetic stirrer (thetime can be varied depending on the scale of the extraction procedure).The supernatant was collected by centrifugation at 5900 Xg for 25 min.at 4° C. At this stage, the extract was stored overnight at -20° C.

EXAMPLE 2 Chromatographic Purification

1. Ion Exchange: Sulfopropyl cellulose ("SP-cellulose") is usedprimarily to concentrate rSP-C prior to size-exclusion chromatography onLH60 resin. SP-cellulose may be prepared by standard methods and thenequilibrated into methanol. It may then be equilibrated in buffer, e.g.,"buffer A" (19:1 isopropanol:0.1 N HCl, plus 50 mM β-mercaptoethanol and0.01 volume NaAc, 2M, pH 4). The organic extract provided in Example 1,at room temperature, is then made 5 mM in HCl by addition of 1Msolution. Resin is added batchwise and incubated overnight at roomtemperature with mixing. SP-cellulose is collected and washed 6×withbuffer A. rSP-C may then be eluted overnight with a small volume (50 mlper liter of cleavage) of a buffer such as "buffer B" (buffer A plus0.025 volumes of NaAc pH 6, 2M) and clarified by filtration throughglass wool.

2. Size-Exclusion Chromatography: LH60 column chromatography removesdimeric and aggregated rSP-C, as well as undesired buffer components,from the monomeric final product. The column was packed with SephadexLH60 obtained directly from Pharmacia. Running solvent was 95%isopropanol, 5% 0.1 N HCl v/v. The column (75 cm in length) was run atroom temperature; a pressure head of 60 cm and a flow rate of 4 ml/cm²per hour.

The volume of sample applied to the column was 3% of bed volume. Elutionof monomeric rSP-C occurred after about 20 hours.

EXAMPLE 3 Hydrophobic Interaction Chromatography Using a CyanopropylColumn

This example describes the use of a cyanopropyl resin to obtain purerSP-C from the entire post-cleavage protein mixture (i.e., withoutprecipitation and extraction) using isopropanol:water solutions to elutethe protein.

Isopropanol, guanidine hydrochloride and DTT were added to thepost-cleavage mixture of Example 1 to give final concentrations of 35%isopropanol, 6M guanidine hydrochloride, and 50 mM DTT. This was appliedto a cyanopropyl column (4.6 mm×3 cm; resin obtained from AppliedBiosystems, Foster City, Calif.). The column was then developed in 5 mMHCl with an isopropanol gradient from 35% to 95% at a flow rate ofapproximately 0.5 ml/min. Protein impurities eluted early in thegradient, while the rSP-C eluted much later, as determined by the UVabsorbance at 228 nm. Western blot analysis confirmed these results.Substantially pure (>90%) rSP-C was thus obtained from the entirepost-cleavage protein mixture.

EXAMPLE 4 Reversed-Phase HPLC

In this example, the use of reversed-phase resins to separate, purifyand assay for rSP-C is described. The method has been found to be usefulfor separating rSP-C monomer from a protein mixture which includes therSP-C dimer.

A mixture of rSP-C monomer and dimer, approximately 95% pure, wasobtained from the LH60 column of Example 2 in isopropanol:0.1N HCl(19:1). The sample was diluted with 1 part 5 mM HCl, injected directlyonto a reversed-phase HPLC C8 column (Brownlee Cartridge, 2.1 mm×3 cm).The column was developed in the presence of 5 mM HCl, with a 40-mingradient from 20%-90% isopropanol. The flow rate used was approximately0.5 ml/min, and the eluted rSP-C monomer was detected via UV absorbanceat 228 nm (Spectroflow 757 Detector). The chart recorder speed was 2mm/min. Virtually complete separation of the monomer from the mixturecontaining the dimer was observed, as confirmed by UV and SDS-PAGE.

EXAMPLE 5

The method of the preceding example was followed, except that prior toinjection of the sample onto the HPLC column, the sample was dried in aSpeed-Vac and dissolved in 95% isopropanol with 50 mM HCl and dilutedwith 50 mM HCl to give a final concentration of 50% isopropanol.Substantially the same results as in the preceding example wereobtained, except that a somewhat cleaner background was obtained here.

EXAMPLE 6

The method of Example 4 was used with a 25-min, 35%-75% gradient of thebuffer solution described. Substantially the same results as in Example5 were obtained.

EXAMPLE 7

The method of Example 4 was used with a 40-min, 35%-95% gradient of thebuffer solution described. Substantially the same results as in Example5 were obtained.

EXAMPLE 8

The procedure of Example 5 is repeated using a reversed-phase HPLC C4column. Substantially the same results as set forth in Example 5 wereobtained.

EXAMPLE 9

The procedure of Example 5 was followed, except a reversed-phase HPLCphenyl column (4.6 mm×3 cm, Applied Biosystems, Foster City, Calif.) wasused. Substantially the same results as set forth in Example 5 wereobtained.

We claim:
 1. A process for separating rSP-C monomer from a mixture ofrSP-C monomer and rSP-C multimers, comprising purifying the mixture byhydrophobic interaction chromatography using an eluant comprising a C₁-C₄ aliphatic alcohol.
 2. The process of claim 1 wherein the C₁ -C₄aliphatic alcohol is isopropanol.
 3. The process of claim 2, wherein theeluant further comprises about 0.001-100 mM acid.
 4. A process forseparating rSP-C monomer from a mixture containing rSP-C monomer andrSP-C multimers, comprising purifying the mixture by reversed-phase highperformance liquid chromatography using a cyanopropyl column and aneluant comprising a C₁ -C₄ aliphatic alcohol.
 5. The process of claim 4,wherein the C₁ -C₄ aliphatic alcohol is isopropanol.
 6. The process ofclaim 5, wherein the eluant further comprises about 0.001-100 mM acidselected from the group consisting of hydrochloric and trifluoroaceticacids.